Enzymatic Synthesis Of Alpha-Galactooligosaccharides And Evaluation Of Its Physiological Function And Structure-Activity Relationship

Galactooliosaccharides(GOS)are typical functional oligosaccharides that comprised of 2-10 monosaccharide units with galactoside linkages.A variety of beneficial health effects have been reported for this group of compounds,such as regulating gut microflora,reducing the risk of cancer,enhancing cardiovascular function,improving immune activities,maintaining urinary tract health,modulating inflammatory response,lowering blood pressure and blood lipids,resisting bacterial and virus and preventing osteoporosis.It has become a mature technology for commercial production of ?-GOS by enzymatic synthesis,however,the study of enzymatic synthesis of ?-GOS is much less than that of ?-GOS.Currently,most of the well documented ?-GOS are present in natural legumes or Chinese herbal medicine.Thus,most of the existing studies of ?-GOS use ?-GOS mixture extracted from those natural materials,which lack the inquiry of their structure-activity relationship.Therefore,a novel structured ?-GOS were obtained by enzymatic synthesis,separation,purification and structure identification in this study.Both the newly synthesized ?-GOS and the ?-GOS extracted from natural materials were used as subjects to evaluate their gut microbiota regulation effects,immunomodulatory activities,anti-inflammatory effects and metabolic syndrome modulation activities.This study was aimed to provide a theoretical basis for further study of the physiological activity of ?-GOS and their structure-activity relationship.The specific research contents are as follows:1.Enzymatic synthesis and structural identification of ?-GOSIn the enzymatic synthesis reaction,90%of the supersaturated galactose solution was used as the substrate and a-galactosidase derived from Aspergillus niger DS was used as the catalyst to synthesize a-GOS under certain conditions.The reaction products of U1,U2,U3 and U4 were detected by High performance liquid chromatography(HPLC)as well as Refractive index detector(RID).The four kinds of reaction products were separated and purified by medium pressure preparative chromatography combined with activated carbon-diatomite filler and normal pressure chromatography combined with polyacrylamide gel.By Electrospray ionization time-of-flight mass spectrometry(ESI-TOF-MS)analysis,U1 and U2 were characterized as disaccharide,U3 and U4 were characterized as trisaccharide.By Nuclear magnetic resonance(NMR)identification,U2 was identified as ?-(1?6)Gal2,U4 was identified as ?-(1?6)Gal3,however,U1 and U3 were identified as mixtures of various compounds.Further study was carried out by 1-phenyl-3-methyl-5-pyrazolone derivatization method combined with HPLC and Diode array detector(DAD)analysis,Ul was speculated as mixture of ?-(1?1)Gal2,?-(1?2)Gal2,?-(1?3)Gal2 and ?-(1?4)Gal2;U3 was speculated as mixture of galacto-trisaccharides linked by one ?-(1?6)glycosidic linkage and one other ?-glycosidic linkage.At the same time,the reaction conditions were optimized and the results showed that the yield of ?-GOS was maximized at conditions of:90%galactose solution as substrate,reaction temperature of 60 ?,enzyme dosage greater than 30 UM/g-galactose,reaction pH of 3.5-5.5.2.Study of regulation effects on gut microflora of ?-GOS with different structuresIn this study,the newly synthesized ?-GOS(Ul,U2,U35 U4 and their mixture ?-GOSg)as well as the ?-GOS extracted from natural materials(mannotriose,stachyose,ciceritol,verbascose)were used as subjects.The effects of ?-GOS with different structures on intestinal microflora were evaluated by fecal anaerobic fermentation in vitro and Fluorescence in situ hybridization(FISH),the secretion of Short-chain fatty acids(SCFAs)were also analyzed by HPLC.The results showed that all ?-GOS treatments significantly promoted the proliferation of total bacteria,Bifidobacterium spp.,Lactobacillus/Enterococcus spp.and Bacteroides-Prevotella spp.Meanwhile,all a-GOS treatments showed no significant promotion effects on Clostridium histolyticum and Clostridium coccoides-Eubacterium rectale,except for verbascose.All ?-GOS treatments significantly promoted the secretion of lactic,acetic,propionic,butyric acids and total SCFAs.At 24 h after anaerobic fermentation,the number of microbial proliferation reached the maximum,thus,the differences between the different ?-GOS treatments were compared at this point.The effects of different structures of a-GOS on Bifidobacterium spp.proliferation were:verbascose>U4>U3 = stachyose>U2 = ciceritol>U1 = mannotriose.The effects of different structures of a-GOS on Lactobacillus/Enterococcus spp.proliferation were:verbascose>stachyose>U4>ciceritol = U2>U3 = mannotriose>U1.These results indicate that in the same structure,?-GOS with high degree of polymerization showed better effects in promotion of Bifidobacterium spp.and Lactobacillus/Enterococcus spp.growth;in the case of the same degree of polymerization,the a-GOS linked only by galactose are more effective than the those containing glucose or fructose;?-GOS linked with ?-(1?6)galactoside linkage showed more effective on the proliferation of probiotics.At the same time,the ?-GOS extracted from natural products showed more significant effect on the promotion of lactic acid,while the ?-GOS obtained by enzymatic synthesis promoted more acetic and butyric acid.3.Immunomodulatory effects of ?-GOS with different structuresIn this study,the newly synthesized ?-GOS(U1,U2,U3,U4 and their mixture?-GOSg)as well as the ?-GOS extracted from natural materials(mannotriose,stachyose,ciceritol,verbascose)were used as subjects.The immunomodulatory effects of ?-GOS with different structures were investigated by culturing mouse macrophage RAW 264.7 in vitro.The results indicated that all ?-GOS treatments significantly promoted macrophage phagocytosis and promoted the secretion of NO,TNF-?,IL-1? and IFN-? cytokines,this effects also showed a dose-dependent manner in concentrations between 25-200 ?g/mL.By comparing the immunoregulatory effects of different structures of ?-GOS,it is shown to a certain extent that ?-GOS linked with ?-(1?6)galactoside linkage were more effective,on this basis,higher degree of polymerization showed better immune regulation activity.4.Anti-inflammatory effects of ?-GOS on colitisThe purpose of this study was to investigate the preventive effects of the newly synthesized?-GOS mixture(?-GOSg),as well as raffinose family oligosaccharides(RFOs),on dextran sulfate sodium(DSS)-induced colitis in mice.The results showed that both ?-GOSg and RFOs alleviated body weight loss,significantly decreased fecal hemoglobin content and prevented colon length shortening.Both ?-GOSg and RFOs inhibited the intestinal mucosal ulcer caused by DSS through histopathological analysis,while ?-GOSg appeared better effects than RFOs.By quantitative Polymerase chain reaction(qPCR)analysis,both ?-GOSg and RFOs attenuated DSS-induced upregulation of COX-2,IL-6 and CSF-1.In addition,Immunohistochemically staining showed that ?-GOSg and RFOs inhibited the expression of nuclear factor(NF-?B)in the colon,however,only ?-GOSg showed statistically significant compared to model group.The results showed that both ?-GOSg and RFOs played the role of resistance to colitis by inhibiting the expression of NF-?B.Meanwhile,?-GOSg have higher anti-inflammatory activities than RFOs in this colitis model.5.Regulatory effects of a-GOS on metabolic syndromeIn this study,a high-fat/westem-style diet(HFWD)was used to induce metabolic syndrome mice by consistently feeding the mice for 13 weeks,the regulatory effects of a-GOSg and RFOs on metabolic syndrome were investigated on this model.The results showed that ?-GOSg significantly inhibited HFWD-induced body weight gain.Through physiological and biochemical analysis,both a-GOSg and RFOs significantly reduced body fat,serum levels of total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and alanine aminotransferase(ALT).However,there were no significant effects on blood glucose level,insulin concentration and insulin resistance upon ?-GOSg and RFOs treatments.Histological analysis of liver samples revealed decreased lipid accumulation in hepatocytes of mice treated with a-GOSg and RFOs.As results,both ?-GOSg and RFOs showed inhibitory effect on metabolic disorders in obese mice.Among them,a-GOSg had better effects than RFOs in various parameters,which suggested that ?-GOSg were more effective on metabolic syndrome.Through qPCR analysis,?-GOSg significantly down-regulated the HFWD-induced lipid metabolic gene Acetyl CoA Carboxylase(ACC)-? up-regulation.Meanwhile,?-GOSg treatment significantly reduced the content of small intestine bile acid,significantly increased the number of Bifidobacterium spp.and decreased the number of Clostridium leptum group.The results indicating that the inhibition of metabolic syndrome may be through the promotion of probiotics proliferation in intestine,and further promote the degradation of bile acid in the distal intestinal.At the same time,?-GOSg may also alleviate the obesity by affecting the de novo fatty acid biosynthesis process.